Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD OF MODULATING THE ACTIVITY OF CALCIUM CHANNELS
IN CARDIAC CELLS AND REAGENTS THEREFOR
FIELD OF THE INVENTION
The present invention relates generally to novel peptides that are capable of
modulating the
activity of calcium channels in cardiac cells. More specifically, the present
invention
provides a method of modulating the activity of a cardiac calcium channel
comprising
contacting a cardiac ryanodine receptor (RyR2) with an amount of a fragment of
a
dihydropyridine receptor (DHPR) polypeptide sufficient to modulate the
activity of said
RyR2, and determining the activity of said calcium channel. The inventive
method is
useful for the treatment of a range of disorders and diseases associated with
cardiac
dysfunction, particularly those diseases and disorders involving reduced
cardiac output
and/or aberrant excitation-contraction coupling, calcium overload, or calcium
leakage, in
cardiac cells.
BACKGROUND TO THE INVENTION
Bibliographic details of the publications referred to in this specification
are collected at the
end of the description. Reference herein to prior art, including any one or
more prior art
documents, is not to be taken as an acknowledgment, or suggestion, that said
prior art is
common general knowledge in Australia or forms a part crf the common general
knowledge
in Australia.
Excitation-contraction coupling is essential to the functioning of striated
muscles, such as
cardiac and skeletal muscles, linking electrical excitation to mechanical
activity. I~ey
components of excitation-contraction coupling are the dihydropyridine receptor
(DHPR), a
voltage-dependent Ca2+ channel of the transverse tubule (TT), and the Ca2+
release
channel or ryanodine receptor (RyR) of the sarcoplasmic reticulum (SR)
membrane that
opens to release calcium from the SR into the cytoplasm.
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Different isoforms of both RyRs and DHPRs exist in cardiac and skeletal muscle
cells. In
particular, the RyRl and DHPR-isoform 3 are predominant in skeletal muscle and
the
RyR2 and DHPR- isoform 1 are predominant in cardiac cells. There is only about
70%
identity between RyRI and RyR2 amino acid sequences.
In skeletal muscle, it is known that the ryanodine receptor (RyRl) is
activated by a protein-
protein interaction with a 138 amino acid cytoplasmic loop between repeats II
and :III ~f
the DHPR oc-1 subunit (Tanabe et al., 1990, Nature 346:507-~~8). A region of
the skeletal
DHPR cytoplasmic loop that is sufficient to activate skeletal muscle RyRI-
mediated
calcium release has been determined to reside within 20 ar~~o acids from
Thr6~l to Leu69o
(El Hayek et al., 1995, J. Biol. Chem. 270:22116-22118; Dulhznty ~t al., 1999,
Bio~ahys. J.
77:189-203; and Gurrola et al., 1999, J. Biol. Chem. 27:7879-7886). There are
four
DHPR molecules located in a tetrad configuration of DHPRs opposite every
second RyRl
polypeptide in skeletal muscle, in a strict geometrical alignment that is
considered to be
important for normal excitation-contraction coupling. During excitation of the
DHPR, such
as by electrical stimulation, this protein-protein interaction presumably
induces a
conformational shift in the RyRl polypeptide that results in channel opening,
thereby
causing calcium efflux from the SR. Accordingly, excitation-contraction
coupling h~a
skeletal muscle is essentially a calcium-independent process.
In contrast, excitation-contraction coupling in cardiac muscle involves a
calcium-i~vduced
calcium release (CICR) mechanism (Niggli, 1999, A~~zu ~e~~ .Phy~iraT, 61:311-
335), T:lxe
opening of cardiac DHPR calcium channels following their excitation results in
a ~rnall
amount of extracellular calcium influx into cardiac myocytes via tl~e voltage-
dependent L-
type calcium channels (i.e. cardiac DHPRs) that are activated during each
action potential.
This initial signal acts as a trigger for subsequent CICR from the
intracellular calcium
stores in the SR. The secondary calcium release from the SR occurs via the
cardiac RyR2
calcium release channels. In most mammals, CICR amplifies the initial signal
trigger
several-fold, consistent with the stoichiometry of cardiac RyR2 molecules to
cardiac
DHPR of about 16:1.
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Notwithstanding that cardiac CICR requires an initial elevation of cytosolic
Ca2+ to trigger
calcium release from the SR, it does not appear to become self sustaining.
This is because
CICR possibly propagates only between cardiac myocytes that are overloaded
with
calcium, however is normally localized to individual cells. Additionally, the
farce of
cardiac muscle contraction increases proportionately with [Ca2+]; between
about i~ x 10-~M
[Ca2+]; and about 10-6M [Caa+]I, suggesting that CICR is not an all or nothing
response.
In contrast to excitation-contraction coupling in skeletal myocytes, it is not
knov~n. ~vbe~ther
or not there is any direct protein-protein interaction between cardiac DHPR
and cardiac.
RyR2 i~ vivo, however the cytoplasmic loop of the skeletal DHPR a-1 subunit
does b ind
to cardiac RyR2 in two hybrid assays (Osterland et al., 1999, Biophys. J.
76:A467-
(abstract)) and the 20-mer peptide (i.e. Thrb~l to IJeu69o) of the skeletal
DHPR a-1 subunit
cytoplasmic loop binds to RyR2 in surface pla~mc~n resonance studies (O'Reilly
and
Ronjat, 1999, Biophys. J. 76:A466-(abstract)).
Compelling data indicate that the relationship between skeletal and cardiac
DHPRs and
RyRs channels is different. For example, cardiac RyR2 expressed in dyspedic
myocytes
that lack skeletal RyRl but contain skeletal DHPR oc-1 subunit, cannot support
skeletal
type excitation-contraction coupling. Additiorjally, isolated RyR2 channels
are not
activated by the entire 138-amino acid cytaplasrnic loop between repeats II
and III of
cardiac or skeletal DHPR a-1 subunits (Lai ~t al., 1994, J. Biol. Claem.
269:6511-X516).
Additionally., the 20-mer peptide (i.e. Thr'~~I to Leu69o) of the skeletal
DHPR a-~1 su'bunit
cytoplasmic loop has been shown not to induce Ca2+ release from cardiac SR, or
to
enhance [3H]ryanodine binding to cardiac RyR2 channels (El Hayelc, et al.,
1995, .s~~p~a),
despite the fact that it is a high affinity activator of skeletal muscle RyRl
chaa~~els.
Moreover, these available data suggest that the 20-mer peptide (i.e. Thrb~l to
Len&~°) of the
skeletal DHPR oc-1 subunit cytoplasmic loop cannot activate cardiac RyR2
channels, such
as, for example, by prolonging their open time or frequency of opening.
Stern (1992, FASEB J. 6:3092-3100) proposed that Ca2+ synapses (i.e. localized
domains
of very ugh Ca2+ near the site of Ca2+ entry and release) functionally link
the activities of
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DHPRs and RyRs in cardiac tissue. Each Ca2+ synapse allows local control of
the RyR2
by virtue of the high local Ca2+ concentration, so as to produce the observed
high signal
amplification without spreading of the Ca2+ release signal across the entire
cell or be~sueen
cells. As a consequence, the number of release units recruited during each
signal could be
quantitatively graded with the trigger calcium release. This proposal is
consistent with the
observation of calcium sparks of short duration (about JO ms) and limited
spatial spread
(about 1.5 Vim) during stimulation of cardiac myezcytes (Cheng et al., 1993,
Science
262:740-744).
Myocardial contractile failure is a common cause of n~crbidity and mortality
in patients
with ischemic heart disease, congestive heart failure, and systemic
inflammatory states
such as sepsis. Accumulating evidence indicates that contractile failure is
associated with
dysregulation of myoplasmic calcium flux. Reduced Ca2~'~ sensitivity of the
myofilaments
or a deterioration of calcium signalling, such as, for example, by
deterioration or disruption
of the calcium synapse, deterioration of the RyR2, or deterioration of the
DHPR, may lead
to a decline in the force of cardiac contractions. Several Ca2+ signal
pathways are adversely
affected during cardiac failure or cardiac hypertrophy (with calcium overload
observed in
end-stage heart failure). An elevated resting Ca'+ concentration, reduced Ca'~
transient
amplitude, slowed relaxation, and altered Ca2~ pzzmp irz the SR have been
observed in
failing or hypertrophic cardiac tissue.
More particularly, hypertrophied as well as failing hearts show decreased
excitation-
contraction coupling efficiencies compared to rzozxnal. hearts (Gomez e~ al.,
1997, Scien~:e
276:755-756). However, individual DHPRs and R~Rs in failing or hypertrophic
hearts
appear normal, suggesting that the link between tlaese two calcium signal
proteins may be
defective. This view is supported by the restoration of normal excitation-
contraction
coupling in hypertrophic cells or following congestive heart failure by the
application of (3-
adrenergic agonists to prolong the open time of cardiac DHPRs.
Additionally, hyperphosphorylation of RyR2 in failing human hearts results in
defective
channel function.
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In addition, cardiac output can be boosted in acute situations, such as after
heart attaclc, by
"inotropic agents" that enhance excitation-contraction coupling. Discrete
areas of heart
muscle are damaged during ischaemic episodes, causing reduced cardiac output.
~lc~c~d
supply to essential organs such as the brain can be maintained by asking the
remaining
healthy heart muscle to contract more strongly. This is currently done by
drugs which
mimic beta adrenergic stimulation and increase cAMP levels to stimulate DHPR
activity
and excitation-contraction coupling. In the long term increased'cAMP levels
can become
toxic and lead to calcium overload.
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SUMMARY OF THE INVENTION
This specification contains nucleotide and amino acid sequence information
prepared using
the program PatentIn Version 3.1, presented herein after the bibliography.
Each nucleotide
or amino acid sequence is identified in the sequence listing by the numeric
indicator <210>
followed by the sequence identifier (e.g. <210>1, <210>2, etc). The length,
type of
sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or
amino
acid sequence are indicated by information provided in the numeric indicator
fields <2I 1>,
<212> and <213>, respectively. Nucleotide and amino acid sequences referred to
in the
specification are defined by descriptor "SEQ ID NO:" followed by the numeric
identifier.
For example, SEQ ID NO: 1 refers to the information provided in the numeric
indicator
field designated <400> 1, etc.
Reference herein to the consensus sequence set forth in SEQ ID N~: 1 shall be
taken to
include a reference to any one or more of the amino acid sequences set forth
in SEQ ID
Nos: 2-7 used to compile said consensus sequence.
Throughout this specification, unless the context requires otherwise, the
wword "comprise",
or variations such as "comprises" or "comprising", will be tmderstood to imply
the
inclusion of a stated step or element or integer or group of steps oz'
elements or integers but
not the exclusion of any other step or element or integer or group of elements
or integers.
As used herein the term "derived from" shall be taken to indicate that a
specified integer
may be obtained from a particular source albeit not necessarily directly from
that source.
In work leading up to the present invention, the inventors sought to identify
novel means
for modulating CICR in cardiac tissue, so as to provide for improved treatment
regimes for
cardiac failure andlor cardiac hypertrophy. Surprisingly, although no physical
connection
between cardiac DHPRs and cardiac RyRs has been established, the present
invention
provides small fragments of the skeletal or cardiac DHPR polypeptides, such
as, for
example, small basic charged peptides, that can activate cardiac RyR2
channels.
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More particularly, the present inventors have shown that the 20-mer peptide
(i.e. TI~r6~1 to
Leu69o) of the skeletal DHPR a-1 subunit cytoplasmic loop produces significant
activation
of RyR2 channels at -40mV (negative potentials induce calcium release from the
SRS and
strong inhibition at +40mV. Activation of cardiac RyR2 was observed at
concentrations as
low as 1nM peptide, significantly less than the peptide concentration required
to activate
skeletal muscle RyRl. Additionally, the inhibition of cardiac RyR2 channels
was
significantly greater than that observed for skeletal RyRl channels at 3 x 10-
~M
cytoplasmic Ca2~.
Accordingly, one aspect of the present invention provides a method for
modulating the
activity of a cardiac ryanodine receptor (RyR2) calcium channel comprising
contacting a
cardiac RyR2 with an amount of a fragment of a dihydropyridine receptor (DHPR)
polypeptide, such as, for example, a basic charged fragment, sufficient to
modulate the
activity of said RyR2.
More preferably, the present invention provides a method for modulating the
activity of a
cardiac ryanodine receptor (RyR2) calcium channel comprising contacting a
cardiac RyR2
with an amount of a fragment of a dihydropyridine ~°eceptor (DHPR)
polypeptide, such as,
for example, a basic charged fragment. sufficient to modulate the acti~~:kty
of said RyR2,
and determining the activity of said cardiac RyR2 calcium channel.
It will be appaxent from the preceding discussion that one embodiment of the
invention is
directed to a method fag enhancing the activity of a cardiac RyR2 calcium
channel
comprising contacting a cardiac RyR2 with an amount of a fragment of a
dihydropyridine
receptor (DHPR) polypeptide sufficient to enhance the activity of said RyR2,
and
determining the activity of said cardiac RyR2 calcium channel. As exemplified
herein and
without limiting the invention to any theory or mode of action or effective
peptide
concentration, the present inventors have shown that, for isolated cardiac
RyR2 in a lipid
bilayer, both the frequency of channel openings and the duration of each
channel opening
is enhanced by the application of up to about 1nM peptide to about IO~IM
peptide. At high
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peptide concentrations, the activity of channels that opened first declines
slightly however
the activity of other channels is more pronounced, presumably reflecting a
microheterogeneity in RyR2 channel sensitivities to peptide.
It will also be apparent from the preceding discussion that another
embodimea~t of the
invention is directed to a method for inhibiting the activity of a cardiac
RyR2 calcium
channel comprising contacting a cardiac RyR2 with an amount of a fragment of a
dihydropyridine receptor (DHPR) polypeptide sufficient to inhibit the activity
of said
RyR2, and determining that said cardiac RyR2 calcium cha~u~el lacks activity.
A.s
exemplified herein and without limiting the invention to any theory or mode of
aCtic~r~ or
effective peptide concentration, the present inventors have s9~.ow~ that, for
isolated cardiac
RyR2 in a lipid bilayer, the frequency of channel openings particularly at
+40mV~ is
reduced by a concentration in excess of about l0~arvl peptide, possibly as a
consequence of
peptide binding within the pore of the RyR2 channel at a site that is distinct
from the site at
which it binds during channel activation.
A second aspect of the invention provides a method for identifying a peptide
modulator of
a cardiac RyR2 calcium channel comprising:
(l) incubating an amount of a fra.gtrae~~t of a dihydropyridine receptor
polypeptide or a homologue, analogue or derivative thereof that ra~od~~lates
cardiac RyR2 channel activity in the presence ~af a functional cardiac RyR2
calcium channel under conditions appropria a for calcium channel activity
to be modulated and determining the activit~~ ~f the channel;
(ii) incubating a candidate peptide in the presence of said functional cardiac
RyR2 calcium channel under conditions appropriate for calcium channel
activity to be modulated by said dihydropyridine receptor polypeptide or a
homologue, analogue or derivative thereof and determining the activity of
the channel;
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(iii) comparing the activity at (i) and (ii); and
(iv) preferably selecting those peptides having comparable or enhanced
modulation of channel activity at (ii) relative to (i).
In an alternative embodiment, this aspect of the invention provides a method
for
identifying a peptide modulator of a cardiac RyR2 calcium channel comprising:
(i) incubating an amount of a fragment of a dihydropyridine receptor
polypeptide or a homologue, analogue or derivative thereof that modulates
cardiac RyR2 channel activity in the presence of a functional cardiac RyR
calcium channel under conditions appropriate for calcium channel activity
to be modulated and determining the activity of the channel;
(ii) incubating a candidate peptide and an amount of said dihydropyridine
receptor polypeptide or a homologue, analogue or derivative thereof that
modulates cardiac RyR2 channel activity in the presence of a functional
cardiac RyR2 calcium channel order conditions appropriate for calcium
channel activity to be modulated by said dihydropyridine receptor
polypeptide or a homologue, analogue or derivative thereof and ~Ietermining
the activity of the channel;
(iii) comparing the activity at (i) and (ii); and
(iv) preferably selecting those peptides having comparable or enhanced
modulation of channel activity at (ii) relative to (i).
A third aspect of the present invention provides a method of determining
whether a cardiac
RyR2 channel is open or has a high channel open probability said method
comprising
contacting a cardiac RyR2 channel with an amount of a fragment of a
dihydropyridine
receptor (DHPR) polypeptide or homologue, analogue or derivative for a time
and under
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conditions sufficient for binding to said channel to occur and determining the
binding of
said peptide to said channel, wherein binding of said peptide to said channel
indicates a
high channel open probability and wherein non-specific peptide binding
indicates a low
channel open probability.
A fourth aspect of the present invention provides a method of treatment of
cardiac
dysfunction in a human or animal subject comprising administering an effective
amrau~~t of
a fragment of a dihydropyridine receptor (DHPR) polypeptide or homologue,
anal~sgue or
derivative for a time and under conditions sufficient far enhanced cardiac
contraction to
occur thereby rectifying said cardiac dysfunction.
A fifth aspect of the present invention provir~es a pharaanaccutical
composition comprising
a fragment of a dihydropyridine receptor polypeptide, which peptide comprises
at least
about 5 contiguous amino acid residues of the peptide set forth in any one of
SEQ ID
NQs: 1-10 or a homologue, analogue or derivative thereof together with one or
more
pharmaceutically acceptable diluents.
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Single and three letter abbreviations used throughout the specification are
defined in
Table 1.
TABLE 1
Single and three letter amino acid abbreviations
Amino Acid Three-letter One-letter
Abbreviation Symbol
Alanine Ala A
Arginine ~'g
Asparagine Asn
Aspartic acid Asp
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine
Leu L
Lysine Lys
Methionine Met M
Phenylalanine Phe
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr
Valine Val V
Any residue or as otherwiseXaa
defined
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation showing the aligned amino acid
seq~.~cnces of the
cytoplasmic loops of several cardiac and skeletal DHPR polypeptides, as
follavas: human
skeletal muscle DHPR-3 (SEQ ID NO: 2; Drouet et al., 1993); murine skeletal
r~~xscle
DHPR-3 (SEQ ID NO: 3; Chaudhari, 1992); rabbit skeletal muscle DHPR-3 (S:~~Q
ID NO:
4; Tanabe et al., 1987); rabbit cardiac DHPR-1 (SEQ ID NO: 5); rat cardiac
D~IPR-1 (SEQ
ID NO: 6); and bullfrog skeletal muscle DHPR-3 (SEQ ID NO: 7; Zhou et al.,
1998). A
consensus sequence (SEQ ID NO: 1) is indicated in bold type, based upon a
comparison of
the available sequences. The amino acid sequences of the non-specific peptides
d~aignated
NB (SEQ ID NO: 8) and A1 S (SEQ ID NO: 9) are also indicated.
Figure 2 is a graphical representation showing that a 20-mer peptide of the
skeletal DHPR
cytoplasmic loop (SEQ ID NO: 2) added to the cytoplasmic (cis) face of
isolated cardiac
RyR2 in a lipid bilayer increases the activity of cardiac RyR2 in the presence
of cis 10-~M
Ca2+ when measured at ~OmV. Single channel activity was determined at -40mV,
either
in the absence of added peptide ( Panel A), or in the presence of 65nM peptide
(Panel B),
or 6.S~M peptide (Panel C).
At the left hand side of each panel, downward channel opening is indicated at -
~Orrr~V,
wherein zero current (closed) level is showr.~ by the dotted line "C" and the
rna~~;.:i~num
single channel conductance is shown by the continuous line "O". Two channels
are active
in the bilayer at -40mV after addition of peptide.
At the right hand side of each panel is a graphical representation of all
points during a 30s
recording period. The numbers on the x-axis indicate the amplitude of channel
opening
and the abscissa indicates the proportion of total openings at each amplitude.
Negative
numbers indicate negative (inward) channel currents at -40mV.
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Figure 3 is a graphical representation showing that a 20-mer peptide of the
skeletal DHPR
cytoplasmic loop (SEQ ID NO: 2) added to the cytoplasmic (cis) solution
increases and
then decreases the activity of cardiac RyR2 in the presence of cis 10-~M Ca2+
at +40mV.
Single channel activity was determined at +40mV, either in the absence of
added peptide
(Panel A), or in the presence of 65nM peptide (Panel B), or 32.Sp.M peptide
(Panel C).
At the left hand side of each panel, upward channel opening is indicated at
+40rnV,
wherein zero current (closed) level is shown by the dotted Iine "C" and the
maxir~~um
single channel conductance is shown by the continuous line "O". The channel is
mostly in
the open configuration at 65nM concentration. In contrast, the channel is
mostly in the
closed configuration in the absence of peptide, or at 32.S~.M peptide.
At the right hand side of each panel is a graphical representation of all
points during a 30s
recording period. The numbers on the x-axis indicate the amplitude of channel
opening
and the abscissa indicates the proportion of total openings at each amplitude.
Numbers
indicate channel opening at +40mV.
Figure 4 is a graphical representation showing the average normalized mean
cuent
(abscissa) as a function of peptide concent~~ation (i.e. loglo[peptide (nM)])
in c~~ttoplasmic
solution (x-axis) at both -40mV (Panel A) and at +40mV (Panel B). Normalized
n~eav~.
current (I'plI'c), is the ratio of the mean current in the presence of peptide
(i.e. l'p) tc~ the
mean current in the absence of peptide under control conditions (i.e. 1'c).
The data indicate
average mean current ~SEM. Samples either contained the 20-mer peptide ofthe
skeletal
DHPR cytoplasmic loop set forth in SEQ ID NO: 2 (n=8, ~), or the non-specific
peptide
NB set forth in SEQ ID NO: 8 (n=2,1), or the non-specific peptide A1S set
forth in SEQ
ID NO: 9 (n=3, ~). A normalized mean current of greater than 1.0 indicates
activation of
the cardiac RyR2 channel by peptide. Accordingly, data indicate significant
activation of
cardiac RyR2 channels by up to 10 ~M peptide (SEQ ID NO: 2) at +40mV, or
higher
concentrations at -40mV.
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Figure 5 is a graphical representation of the subject peptides increasing Ca2+-
and caffeine-
activated Ca2+ release from cardiac SR vesicles. The initial rate of Ca2+
release is shown
under control conditions or following addition of either 20 ~.M Ca2+ or with 2
mM caffeine
(at zero peptide concentration) and after addition of peptide alone (small
symbols), or after
addition of 20 ~M Ca2+ or 2 mM caffeine plus peptide (large symbols) to the
e~~ravesicular
solution at the concentrations shown on the abscissa. The rate of Ca2+ release
is given as
p.moles of Ca2+, per mg of protein in the SR .vesicles, per min. Data is shown
for SDQ ID
NO: 2 (filled circles), SEQ ID NO: 8 (filled squares), SEQ ID 1"30: 9 (open
circles) ~cl
SEQ ID NO: 10 (open squaxes). Results are given as mean~sem with at least 5
observations for each concentration.
Figure 6 is a graphical representation of ~:he effects of SEQ ID NO: 9 on the
mean current
flowing through RyR channels incorporated into lipid bilayers, with a cis
(cytoplasmic)
Ca2+ concentration of 100 ~M. Data was obtained from 60s of recordings at a
bilayer
potential of -40 mV (upper graph) and +40 mV (lower graph). The data points
show
average relative mean current (mean current with peptide divided by mean
current under
control conditions) from six experiments and the vertical bars show ~1 sem.
Channel
activity is clearly depressed by the peptide in a concentration-dependent
manner with
maximum effects at 100nM peptide. The depression at 100 nM peptide was
signific.tly
greater at +40 mV (asterisk) than at -40 ra~~l"
Figure 7 is a graphical representation of the effects of SEQ ID NO: 9 on the
mean current
flowing through RyR channels incorporated into lipid bilayers, with a cis
(cytoplasn~ic;~
Ca2+ concentration of 100 nM. Data was obtained from 60s ofrecordings at a
bilayer
potential of -40 mV (upper graph) and +40 mV (lower graph). The data points
show
average relative mean current (mean current with peptide divided by mean
current under
control conditions) from seven experiments and the vertical baxs show ~1 sem.
Channel
activity is clearly enhanced by the peptide in a concentration-dependent
manner with
maximum effects at 100nM peptide. Depression at >100 nM peptide was seen at
+40 mV
and is indicative of some pore block.
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Figure 8 is a graphical representation of the effects of SEQ ID NO: 9 on
single channel
parameters as a function of peptide concentration. Data is shown at -40mV
(left panel) and
+40mV (right panel). The top graphs show the open probability of the channels,
the second
graphs show mean open time, followed by mean closed time and finally the
frequency of
channel opening. The data points show average parameter values from seven
experiments
and the vertical bars show ~1 sem. The decrease in the closed time of the
channel and
consequent increase in frequency contribute most strongly to the increase in
open
probability.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention provides a method for modulating the
activity of a
cardiac ryanodine receptor (RyR2) calcium channel comprising contacting a
cardiac RyR2
channel with an amount of a fragment of a dihydropyridine receptor (DHPR)
polypeptide
sufficient to modulate the activity of said RyR2.
More particularly, the present invention provides a method for z~zodulating
the activity of a
cardiac ryanodine receptor (RyR2) calcium channel comprising contacting a
cardiac RyR2
channel with an amount of a fragment of a dihydropyridine receptor (DHPR)
polypeptide
sufficient to modulate the activity of said RyR2, and detel-rznining ~ze
activity of said
cardiac RyR2 calcium channel.
As used herein, the term "ryanodine receptor-2 channel" or "RyR2 channel" or
"cardiac
RyR channel" shall be taken to refer to a calcium channel that comprises RyR2
polypeptide subunits. Those skilled in the art are aware of the physical
structure of a
"RyR2 channel" or "cardiac RyR channel" referred to herein.
As used herein, the term "modulating" shall be taken to mean enhancing or
inhibiting the
activity of an RyR2 calcium channel. Accordingly, by ''zxzodifying the
activity of arz P.yR2
calcium channel" or similar term is meant generally that C~hCR is modified
(i.e. cxzbced
or reduced) such as, for example, by modifying the calcium synapse or calcium
sensitivity
of cardiac RyR2, the frequency of cardiac RyR2 channel openizngs during each
action
potential, or the open time of individual cardiac RyRs.
As stated sup~cz the present invention clearly encompasses both a method for
ez~ancing the
activity of a cardiac RyR2 calcium channel and a method for inhibiting the
activity of a
cardiac RyR2 calcium channel.
The cardiac RyR2 channel may be in situ in a cardiac cell, or in cardiac
muscle ih vivo,
however it may also be an isolated RyR2 chamiel, such as, for example,
inserted in lipid
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bilayer, or alternatively, a recombinant or reconstituted RyR2 channel such
as, for
example, expressed in the membrane of a transfected cell (e.g. a CHO cell or
dyspedic
myocyte). Standard procedures, such as, for example, as described by I3hat et
al. (1997,
Biophys. J. 73:1329-1336), which is herein incorporated by reference, can be
used to
express the RyR2 channel in transfected cells. Preferably, the cardiac RyI~2
channel is ih
situ in a cardiac cell or in vivo in cardiac tissue.
As used herein, the term "fragment" means that the peptide has an overall
positive charge
at physiological pH values by virtue of the presence of a relatively high
propox°tion of basic
amino acid residues, such as, for example, arginine or lysine, or the half
basic ~-amino acid
residue, histidine. Preferably, a fragment will have at least: about 25% basic
amino acid
residues, and more preferably at least about 50% basic ~nano acid residues.
The peptide that is useful in performing the invention descz~ibed herein is of
at least about 5
amino acids in length from a cardiac or skeletal dihydropyridine receptor
(DHPR)
polypeptide fragment comprising the amino acid sequence TSAQKXXXEE
(R/K)XR(RlK)K(M/L)(A/S)(R/K)XX (SEQ ID NO: 1), wherein X is any amino acid
residue. The present invention clearly extends to the use of peptides having a
similar
sequence to SEQ ID NO: 1 from any source, such as, for example, a synthetic of
naturally-
occurring peptide, or a peptide derived from any DHPR sequence.
For the purposes of further describing the invention, tl~e amino acid sequence
set forth in
SEQ ID NO: 1 corresponds to a 20-mer peptide consensus sequence for the
cytoplasmic II-
III loop of the DHPRs of skeletal and cardiac muscle of several animal
species, as
indicated in Figure 1. As exemplified herein, the present inventors show that
a portion of
the cytoplasmic II-III loop of human skeletal muscle DHPR-3 modulates sheep
RyR2
channel activity. Close sequence relationships between the various DHPRs
listed in Figure
1 indicates that any peptide having the sequence of SEQ ID NO: 1 or
substantially
identical to SEQ ID NO: 1 will modulate RyR2 channel activity.
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Accordingly, the present invention clearly extends to the use of any and all
homologues,
analogues and derivatives of the amino sequence set forth in SEQ ID N0: 1.
Preferably,
such homologues, analogues, or derivatives will be basic charged peptides,
more
preferably retaining the conserved basic residues of SEQ ID NO: 1.
Even more preferably, the subject sequence comprises the motif RKRRK at amino
acid
positions 11 through to 15 of SEQ ID NO: 1.
In the present context, "homologues" of any one of SEQ ID NOs: 1-7 refer to
those natural
or synthetic basic charged peptides that are derived directly front skeletal
or cardiac DHPR
amino acid sequences or similar sequences from other sources, such as other
special, or
alternatively, by screening of mimetic peptides for RyR2 channel modulatory
activity, and
comprise a sequence that corresponds substantially to SEQ ID N0: 1 or any one
of the
specific amino acid sequences listed in SEQ ID Nos: 2-7 inclusive.
For example, amino acids of any one of SEQ ID Nos: 1-7 may be replaced by
other amino
acids having similar properties, for example hydrophobicity, hydrophilicity,
hydrophobic
moment, antigenicity, charge or propensity to form ec-helical structures,
provided that the
overall characteristics (e.g. basic charge or conformation) of the peptide are
maintained.
Substitutions encompass amino acid alterations in which an amino acid is
replaced r~~th a
different naturally-occurring or a non-conventional amino acid residue. Such
substita~tions
may be classified as "conservative", in which case an amino acid residue is
replaced with
another naturally-occurring amino acid of similar character, such as, for
example,
Gly~Ala, VahIle~Leu~Met, Asp~Glu, Lys~Arg, or Asn~Gln. Amixzo acid
substitutions are typically of single residues, but may be of multiple
residues, either
clustered or dispersed.
Homologues that are synthetic peptides produced by any method known to those
skilled in
the art, such as by solid phase methods using Fmoc amino acids in an automated
peptide
synthesizer, are particularly contemplated by the present invention. Such
peptides may be
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subjected to cyclization by conventional procedures, andlor be partially
purified such that
they are substantially free of conspecific peptides.
"Analogues" of any one of SEQ ID NOs: 1-7, encompass those amino acid
sequences that
are substantially identical to said sequences or a homologue thereof,
notwithstanding fhe
occurrence of one or more non-naturally occurring amino acid analogues
therein.
Particularly preferred analogues include any peptide or non-peptide mimetics
of any one of
SEQ ID NOs: 1-7 that retain the characteristics of said sequences, such as,
for example,.
charge distribution or conformation or other structural characteristic. The
imperatoxin
peptide described by Gurrola et al (1999, ,I. Biol. Chem. 274:7879-7880), and
any
synthetic non-peptide mimetics are particularly contemplated by the present
invention.
The term "derivative" in relation to any one of SEQ ID NOs: 1-7 shall be taken
to refer to
any parts, fragments or polypeptide fusions of said sequence or a homologue or
analogue
thereof. Derivatives include modified amino acid sequences or peptides in
which ligands
are attached to one or more of the amino acid residues contained therein, such
as, for
example, a lipid, liposaccharide, lipopolysaccharide (LPS), carbohydrate,
enzyme, peptide,
radionuclide, fluorescent compound, photoactivatable residue (e.g. p-benzoyl-
phenylalanine), or glucosyl moiety. Procedures for derivatizing peptides are
well-known in
the art.
For example, preferred derivatives may comprise a fragment of any one of SEQ
ID NCI~s:
1-7 or a fragment of a homologue or analogue of any one of SEQ ID NOs: 1-7.
Amino
acid deletions will usually be of the order of about 1-15 amino acid residues
in length.
Deletions are preferably made to the N-terminus or C-terminus of SEQ ID NO: 1.
Alternatively, derivatives of any one of SEQ ID NOs: 1-7 may have additional
amino acid
residues added to the N-terminus or the C-terminus of the peptide or a
homologue or
analogue thereof. Insertions will generally be sufficiently small so as to not
hinder access
to the RyR2 channel, such as, for example, insertions of the order of 1-4
amino acid
residues.
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Preferred homologues, analogues and derivatives of any one of SEQ ID NOs: 1-7
will
comprise at least about 5 contiguous amino acids of any one of SEQ ID Nos: 1-
7, more
' preferably at least about 10 contiguous amino acid residues or more
preferably at Least
about 15-20 contiguous amino acid residues. Accordingly, such homologues,
analogues
and derivatives may be full-length or less than full-length sequences compared
to any one
of SEQ ID NOs: 2-7.
In addition to possessing functional equivalence to SEQ ID NO: 1 in so far as
RyR2
channel modulatory activity is concerned, a preferred homologue, analogue or
derivative
will comprise an amino acid sequence having at least about 70% identity to SEQ
ID IdrO: 1.
Preferably, the percentage identity to SEQ ID NO: 1 will be at leapt about
80%, more
preferably at least about 90% and even more preferably at least about 95% or
at least about
98 or 99%. In determining whether or not two amino acid sequences fall within
defined
percentage identity or similarity limits, those skilled in the art will be
aware that it is
necessary to conduct a side-by-side comparison of amino acid sequences. In
such
comparisons or alignments, differences will arise in the positioning of non-
identical amino
acid residues depending upon the algorithm used to perform the alignment. In
the present
context, references to percentage identities and similarities between two or
more amino
acid sequences shall be taken to refer to the number of identical and
sixrgilar residues
respectively, between said sequences as determined using any standard
algorithm known to
those skilled in the art. In particular, amino acid identities and
similarities are calculated
using the GAP program of the Computer Genetics Group, Inc., University
Research Park,
Madison, Wisconsin, United States of America (Devereaux et al, 1984, Nucl.
Acids Res.
12: 387-395), which utilizes the algorithm of Needleman and Wunsch (1970, J.
ll~~l. ~3iol.
48: 443-453) or alternatively, the CLUSTAL W algorithm of Thompson et al
(1994, Nucl.
Acids Res. 22: 4673-4680) for multiple alignments, to maximize the number of
identical/similar amino acids and to minimize the number and/or length of
sequence gaps
in the alignment.
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Particularly preferred homologues, analogues, or derivatives of SEQ ID NO: 1
will
successfully compete with any one of SEQ ID NOs: 2-7 for modulation of cardiac
RyR2
channel activity. In standard competition studies, different concentrations of
the peptide
being tested are assayed for RyR2 channel modulatory activity in the presence
c~f
concentrations of SEQ ID NO: 2, for example, that either enhance or inhibit
cardiac RyR2
channel activity. For example, a high affinity activator of a cardiac RyR2
channel cyan be
determined by its ability to successfully enhance channel activity in a lipid
bilayer at a
cytoplasmic calcium concentration in the range of about 10-~M to about 10-SM,
arid tn
compete for the enhancement of channel activity that is induced by known
activat~aar
peptide (e.g. about 1nM SEQ ID NO: 2 to about 100nM SEQ ID NO: 2). Similarly,
a high
affinity inhibitor of a cardiac RyR2 channel can be determined by its ability
to successfully
inhibit channel activity in a lipid bilayer at a cytcplasmic calcium
concentration in the
range of about 10-~M to about 10'5M, and to e~ampete for the inhibition of
channel activity
that is induced by known a inhibitor peptide at cytoplasmic Ca2+
concentrations up to 10-
4M (e.g. 32.5 ~M SEQ ID NO: 2).
Without limiting the present invention in any way, the inventors have
generated three
peptides which correspond to analogues and derivatives, as defined herein, of
the human
skeletal DHPR 20-mer peptide sequence described in SEQ ID NO: 2. These
peptides are
detailed below:
(i) SEQ ID NO: 8 corresponds to the 20-mer peptide of SEQ ID NO: 2 hut
wherein serine68~ (residue 17 of SEQ ID NO: 2) is replaced by an alanine
residue, as follows:
TSAQKAKAEERI~RRKMARGL (SEQ ID NO: 8)
(ii) SEQ ID NO: 9 corresponds to the 20-mer peptide of SEQ ID NO: 2 but
wherein arginine688 (residue 18 of SEQ ID NO: 2) is replaced by the D
isomer of arginine, as follows:
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TSAQKAI~AAEERI~RRI~MSRDGL (SEQ ID NO: 9)
(iii) SEQ ID NO: 10 corresponds to the 20-mer peptide of SEQ ID NO: 2 but
wherein both serine68~ (residue 17 of SEQ ID NO: 2) is replaced by an
alanine residue and arginine688 (residue 18 of SEQ ID NO: 2) is replaced by
the D isomer of arginine, as follows:
TSAQKAKAEERI~:P.RI~.~vIARDGL (SEQ ID NO: 10)
In the present context, for example, the tern. "contacting" may mean a
caa:diac RyR2
channel with a basic peptide fragment of a DHPR polypeptide is meant that the
peptide is
brought into close physical association with the cha~mel without necessarily
requiring
actual binding of the peptide to the channel. Notwithstanding that the DHPR
peptide mad
bind to the RyR2 channel in performing the invention, such binding is not an
essential
feature of the invention, because all that is required is modified channel
activity. In this
respect, the purpose of the invention is not to achieve binding to the cardiac
RyR2
polypeptide, but to modulate activity of the RyR2 channel. Those skilled in
the art are
aware that binding and activity are not necessarily equivalent, because a DHPR
peptide
may bind to any one of a multitude of different sites on a RyR2 polypeptide
without
necessarily modifying the activity of the RyR2 channel.
Preferably, the peptide is contacted with the cytoplasmic face of the RyR2
channel.
Preferably, any one of SEQ ID NOs: 1-7 or a homologue, analogue, or derivative
of any
one of SEQ ID NOs: 1-7, such as any one of SEQ ID NOS: 8-10, binds to a part
of the
RyR2 polypeptide in the RyR2 channel during performance of the inventive
method.
Without being bound by any theory or mode of action, the peptide (or a
homologue,
analogue, or derivative thereof) used in performing the invention can assume a
conformation that gains access to the RyR2 channel by binding to one or more
negatively
charged residues of the RyR2 polypeptide in the channel, such as, for example,
acidic
residues in the amino acid sequence:
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FRAEKTYAVKAGRWYFEFEAVTSGDMRVGWSRPGCQP (SEQ ID NO: 13).
It follows from the preceding discussion that, to determine activity of the
cardiac RyR2
calcium channel, it is not sufficient to merely measure binding of the
peptide, or ryanodine
for that matter, to the channel or to the RyR2 polypeptide, although that may
certainly
form an adjunct to determining modified channel activity. Preferred means for
determining channel activity are selected from the group consisting of:
(i) recording of single or multiple RyR2 channel openings in lipid bilayers
using art-recognized procedures, such as, for example, those described by
Ahern et al. (1994, FEBS Lett. 352~3~9-374), Lu et al., (1994, J. Biol.
Chem. 269:6511-6516), Laver et al.. (1995, ~ Nlenzbr. Biol. 147:7-22), or
Dulhunty et al. (1999) sups°a;
(ii) determination of calcium release from SR vesicles using art-recognized
procedures, such as, for example, those described by El-Hayek et al. (1995,
supra), Gurrola et al. (1999, supra), or Dulhunty et al. (1999, supra);
(iii) determination of cardiac function, such as, for example, by determining
cardiac contractility according to ~aloga et crl. (1997); and
(iv) determination of vascular tone of isolated thoracic aortic rings
following
peptide administration using any art recognized method for deternc~ining
vascular tone.
Those skilled in the art are aware that cardiac function can be readily
determined by
measuring the concentration dependence of peptide administration on one or
more
parameters selected from the group consisting of contractility as assessed by
dP/dtmax or -
dP/dtmax values, left ventricular systolic pressure, and heart rate.
Ventricular fibrillation or
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other cardiac arrhythmia can also be determined to quantify negative side-
effects of
peptide.
In assaying for enhanced cardiac RyR2 channel activity, an enhanced channel
opening
probability (Po) is detected. The higher channel opening probabilit~,T leads
to enhanced.
calcium efflux or release from the SR, including from SR vesicles, and
enhanced cardiac
contractility, and reduced relaxation. Vascular tone may also be enhanced.
In contrast, inhibitors of cardiac RyR2 channel activity will decrease the
channel open
probability, reduce calcium release from the SR, and reduce cardiac
contractility. Vascular
tone may also be reduced.
Additional methods of determining modified cardiac RyR2 channel activity are
not
excluded.
The efficacy of any one of SEQ ID NOs: 1-7 or a homologue, analogue or
derivative
thereof (such as SEQ ID NOs: 8-10), in particular SEQ ID NO: 2, in modulating
cardiac
RYR channel activity, establishes the utility of such sequences as a reagents
for use in
screening for compounds sharing structural and functional similarity. Such a
reagent
enables a high-throughput screening assay in which thousands of compounds can
be
rapidly screened. Peptide mimetics potentially having the modulatory activity
described
herein can be tested for their ability to modulate cardiac RyR2 channel
activity with
isolated cardiac RyR2 receptors in lipid bilayers, or in another suitable
assay format.
Accordingly, a second aspect of the invention provides a method for
identifying a peptide
or non-peptide modulator of a cardiac RyR2 calcium channel comprising:
(i) incubating an amount of a fragment of a dihydropyridine receptor
polypeptide or a homologue, analogue or derivative thereof that modulates
cardiac RyR2 channel activity in the presence of a functional cardiac RyR2
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calcium channel under conditions appropriate for calcium channel activity
to be modulated and determining the activity of the channel;
(ii) incubating a candidate peptide or non-peptide compound in the presence of
said functional cardiac RyR2 calcium channel under conditions appropriate
for calcium channel activity to be modulated by said dihydropyridine
receptor polypeptide or a homologue, analogue or derivative thereof anel
determining the activity of the channel; and
(iii) comparing the activity at (i) and (ii).
Preferably said fragment comprises at least 5 contigzzous amino acids of tl~e
peptide
sequence of SEQ ID NO: 1.
Preferably, those peptides having comparable or enhanced modulation of channel
activity
to SEQ ID NO: 1 or a homologue, analogue or derivative of SEQ ID NO: 1 are
selected.
Such peptides are detectable by the comparable or enhanced modulation of
channel
activity detected at (ii) relative to (i) supt°a.
For example, a fixed amount of SEQ ID NO: 1 or a homologue, analogue ~or
derivative
thereof which modulates cardiac RyR2 channel activity can be added to the
functional
receptor. The reaction mixture is then incubated under conditions appropriate
for activity
to be modulated, and activity of the channel is determined. In a parallel
experiment, a
candidate peptide or non-peptide compound is incubated with the cardiac
channel under
conditions that permit modulation of activity in the presence of SEQ ID NO: 1
and the
channel activity of this test sample is determined relative the activity of
SEQ ID NO: I or
its homologue, analogue or derivative. Those peptides or non-peptide compounds
having
comparable or enhanced modulatory activity relative to SEQ ID NO: 1 or its
homologue,
analogue or derivative, can be selected.
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In an alternative embodiment, this aspect of the invention provides a method
for
identifying a peptide or non-peptide modulator of a cardiac RyR2 calcium
channel
comprising:
(i) incubating an amount of a fragment of a dihydropyridine receptor
polypeptide or a homologue, analogue or derivative thereof that modulates
cardiac RyR2 channel activity in the presence of a functional ~;ardiac RyR2
calcium channel under conditions appropriate frar calcium cha~:el activity
to be modulated and determining the activity s~f the channel;
(ii) incubating a candidate peptide or non-peptide compound and an amount of
said dihydropyridine receptor polypeptide or a homologue, analogue or
derivative thereof that modulates cardiac RyR2 channel activity m the
presence of a functional cardiac RyR2 calcium channel under conditions
appropriate for calcium channel activity to be modulated by said
dihydropyridine receptor polypeptide or a homologue, analogue or
derivative thereof and determining the activity of the channel; and
(iii) comparing the activity at (i) and (ii).
Preferably, said fragment comprises at least 5 contiguous an°~i~~ac~
acids of the pep~de
sequence of SEQ ID NO: 1.
In a most preferred embodiment of these aspects of the present invention, said
SEQ ID
NO: 1 peptide or homologue or derivative thereof is selected from any one ox
more of SEQ
ID NOs: 2-10.
Preferably, those peptides or non-peptide mimetics etc having comparable or
enhanced
modulation of channel activity to SEQ ID NO: 1 or a homologue, analogue or
derivative of
SEQ ID NO: 1 are selected. Such peptides or non-peptide compounds are
detectable by
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the comparable or enhanced modulation of channel activity detected at (ii)
relative to (i)
supra.
For example, a fixed amount of SEQ ID NO: 1 or a homologue, analogue or
derivative of
SEQ ID NO: 1 that modulates cardiac RyR2 channel activity is added to the
functional
receptor under conditions that permit modulation to occur. The activity of the
channel is
determined in the presence or absence of a candidate peptide and the channel
activities of
the saanples compared. Those peptides or non-peptide compounds that modulate
the effect
of SEQ ID NO: 1, or its homologue, analogue or derivative; on cardiac RyR2
calcium
channel activity can be selected.
Any conventional assay format which relies on modulation of cardiac RyR~,
channel
activity is appropriate for this purpose. In a preferred assay format, the
sample to be tested
is a reconstituted RyR2 channel in a planar lipid bilayer or a SR vesicle or a
dyspedic
myocyte having a functional cardiac RyR2 calcium channel. Using only routine
experimentation, one skilled in the art can determine whether a particular
candidate
peptide, or non-peptide compound, modulates activity of a cardiac calcium
channel.
The present invention clearly contemplates a process that utilizes rapid, high
throughput
screens with some tolerance of non-specificity and/or smaller-scale f-
unction~:~l sereens
having higher specificity, and/or quantitative lcinetic studies to elucidate
chemica.I
structure/function relationships of the cardiac RyR2 channel, such as, for
example, the
determination of peptide or non-peptide compounds that are agonists or
antagonists of
cardiac RyR2 calcium channel function, or the elucidation of the docking
sites) for said
compounds in the channel.
Preferably, the present invention contemplates a process comprising:
(i) identifying candidate agonists and antagonists of a cardiac RyR2 calcium
channel;
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(ii) determining those compounds at (i) that actually activate or inhibit the
activity of a cardiac RyR2 channel;
(iii) determining which compounds at (ii) have higher binding affinities for
said
cardiac RyR2 calcium channel than any one of SEQ ID Nos:l-10; and
(iv) optionally, determining the sites of interaction between those compounds
at
(iii) and said cardiac RyR2 calcium channel.
Rapid, high throughput screens to identify candidate agonists and antagonists
of a cardiac
RyR2 calcium channel are preferably carried out using cardiac RyR2 calcium
channels in
microsomal preparations of cardiac muscle, or alternatively, expressed in CHO
cells or
other suitable cell-based assay system. Such high throughput screens
facilitate the
screening of large numbers of compounds incubated with the microsomal
preparations, or
injected into or incubated with transfected cells expressing the RyR2 channel.
High
throughput screens also facilitate the screening of large numbers of peptides
expressed
from libraries that have been transfected into cells expressing the RyR2
channel.
Alternatively, or in addition, candidate agonist and antagonist molecules are
identified by
binding the cardiac RyR2 protein to a support such as a pharality of polymeric
pins, and
bringing the polypeptide on the plurality of pins into contact with
cafixdidate agonist and/or
antagonist molecules for screening. The molecules being screened may be
isotopically
labelled so as to permit ready detection of binding. Alternatively, compounds
for
screening may be bound to a solid support, such as a plurality of pins which
are reacted
with the RyR2 polypeptide. Binding may, for example, be determined again by
isotopic-
labelling, or by antibody detection, or use of another reporting agent.
The binding affinity of a particular chemical compound for a cardiac RyR2
calcium
channel may be determined by any assay known to those skilled in the art to be
useful for
determining kinetic parameters of protein-Iigand interactions. Preferably, a
binding assay,
such as, for example, surface plasmon resonance, is employed. The surface
plasmon
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resonance of a protein may be determined, for example, using a BiacoreTM
analyzer. As
will be known to those skilled in the art, this method provides data for the
determination o~
on and off rates for the binding of a ligand to a protein of interest.
To screen multiple candidate compounds, the compounds may be attached to a
plurality of
polymeric pins or supports.
To determine the sites) of interaction between a candidate compound and the
cardiac
RyR2 calcium channel, the binding of the candidate compounds to various mutant
RyR2
polypeptides having one or more sites in the native protehz deleted or
substituted with a
variant amino acid sequence can be determined, and ~compa-red to the binding
of said
compound to the wild-type or non-mutant RyR2 protein. Again, surface plasmon
resonance may be employed to facilitate the comparison of binding affinities.
Data on
those sites of interaction which provide stronger aganistlantagonist activity
are used to
facilitate the rational design of drug which bind to such sites, albeit with
enhanced binding
affinities.
Compounds detected using this screening procedure can ultimately be used, for
example,
in the treatment.of cardiac dysfunction.
In an alternative embodiment, agonist and/or antagarais'.t ~coz~npounds are
identified using
rational drug design; by identifying compounds which bind to or associate with
the three-
dimensional structure of the cardiac RyR2 calcium channel. The present
invention clearly
contemplates any synthetic compounds derived from the three dimensional
structure of the
cardiac RyR2 calcium channel that bind to said channel and that activate or
inhibit cardiac
RyR2 calcium channel activity.
The observation that the open probability of a cardiac RyR2 channel is
modified by
incubation with SEQ ID NO: 1 or a homologue, analogue or derivative thereof
establishes
the utility of such sequences in establishing or determining the pore
structure and
regulation mechanism of the cardiac RyR2 channel in vitro and in vivo. More
particular~.y,
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the correlation between binding of the peptide to RyR2 and high open
probability of the
channel on the one hand, and the correlation between non-specific binding of
peptide at
negatively charged residues within the channel pore and low open probability,
makes
possible the prediction of open probability based upon peptide binding
studies.
Accordingly, a third aspect of the present invention provides a method of
determin~t~g
whether a cardiac RyR2 channel is open or has a high channel open probability
said
method comprising contacting a cardiac RyR2 channel with an amount of a
fragment ~xf a
dihydropyridine receptor (DHPR) polypeptide or homologue, analogue or
derivative
thereof for a time and under conditions sufficient for binding to RyR2 to
occur and
determining the binding of said peptide to RyIZ.2, wherein binding of said
peptide to RyR2
indicates a high channel open probability and wherein non-specific peptide
binding of
peptide to the channel pore indicates a low channel open probability.
Preferably, said fragment is a peptide substantially as defined in any one or
more of SEQ
ID NOs: 1-10.
Peptide binding can be determined by any method known to the skilled person,
such as, for
example, by using a radioactive labeled or fluorescent labeled peptide, or
peptide labeled
with a reporter molecule, and determining the amount of label or reporter
molecule bound
to the channel at any particular concentration of peptide. Preferrer3.
reporter molecules f or
this purpose are small molecules that do not hinder the ability of the peptide
to bind tea the
channel, such as, for example, photoactivatable compounds.
Alternatively, peptide binding may be determined indirectly by measuring the
acti:~ita~ of
the channel, of the calcium release through the channel. As exemplified
herein, peptide at
a concentration between about 1nM and about 10 ~M peptide produces a high
channel
open probability, whereas peptide at a higher concentration indicates a low
channel open
probability, particular for channels in lipid bilayers.
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A fourth aspect of the present invention provides a method of treatment of
cardiac
dysfunction in a human or animal subject comprising administering an effective
amount of
a fragment of a dihydropyridine receptor (DHPR) polypeptide or derivative,
homologue or
analogue thereof for a time and under conditions sufficient for enhanced
cardiac
contraction to occur thereby rectifying said cardiac dysfunction.
Preferably, said fragment comprises at least 5 contiguous amino acids of a
pcp~:ide
substantially as defined in any one or more of SEQ ID I'~I~3s: 1-10.
By ''cardiac dysfunction" is meant a condition involving impaired myocardial
contraction,
such as, for example, wherein Ca2+ sensitivity of the myofilaments is reduced,
or there is a
deterioration of calcium signalling, such as, for example, by deterioration or
disruption of
the calcium synapse, deterioration of the RyR2, or deterioration of the DHPR.
Ct~nditions
of cardiac dysfunction contemplated herein to be amenable to treatment
according to the
present invention include, but are not limited to, myocardial contractile
failure, ischemic
heart disease, systemic inflammatory states such as sepsis, cardiac
hypertrophy (calcium
overload), cardiomyopathy such as arrhythmogenic right ventricular dysplasia
type-2
(ARVD2), and drug (e.g. cocaine)-induced cardiomyopathy, infarction,
dysrhythmia,
congestive heart failure, or heart attack.
By "effective amount" means an amount of a peptide suf~c.ievt to diminish or
reverse
progression of the dysfunction.
For purposes of this aspect of the invention, beneficial or desired clinical
results include,
but are not limited to, alleviation of symptoms, diminishment of extent of
disease,
stabilization of the disease state, delay or slowing of disease progression,
amelioration or
palliation of the disease state, and remission (whether partial or total),
whether detectable
or undetectable. "Treatment" also includes prolonging survival as compared to
the
expected survival of a subject not receiving treatment. As used herein, the
term "treatment '
includes prophylaxis.
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"Palliating" a disease means that the extent and/or undesirable clinical
manifestations of a
disease state are lessened and/or the time course of the progression is slowed
or
lengthened, by treatment.
In the context of prophylaxis, a "subject" includes, but is not limited to,
individuals in the
general population who are about 40 years of age or older; and, in particular,
an individual
with a history or predisposition to developing cardiac hypertrophy, cardiac
myopathy,
heart attack, hypertension, renal failure, vascular hypertension, respiratory
ailment such as
emphysema or cystic fibrosis, chronic asthma, and tuberculosis. Suitable
subjects also
include organ transplant patients.
In a fifth aspect of the present invention there is provided the use of a
fragment of a
dihydropyridine receptor polypeptide comprising at least 5 contiguous amino
acid residues
of the peptide set forth in any one of SEQ ID NOs: 1-10 or a homologue,
analogue or
derivative thereof to modify the activity of a cardiac ryanodine calcium
channel, thereby
modifying defective calcium signaling.
Preferably, said defective calcium signaling induces chronic hypertrophy,
dilated cardiac
myopathy or heart failure.
In a sixth aspect of the present invention there is provided the use of a
fxa~nent of a
dihydropyridine receptor polypeptide comprising at least 5 contiguous amino
acid residues
of the peptide set forth in any one of SEQ ID NOs: 1-10 or a homologue,
analogue or
derivative thereof in the manufacture of a medicament for the treatment of
cardiac
dysfunction in a human or animal subject.
This invention involves the use of a peptide set forth in any one of SEQ ID
Nos: 1-10 or a
homologue, analogue, or derivative thereof, to modify the activity of cardiac
RyR2
calcium channel, thereby modifying defective calcium signaling that induces
chronic
hypertrophy or dilated cardiac myopathy or even heart failure in the chronic
untreated
animal of human subject.
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Preferably, the peptide or homologue, analogue or derivative thereof is
administered at a
dosage that can enhance contractile force, and further increase intracellular
calcium
concentration (i.e. [Ca2+];) during systole, and further decrease [Caa+];
during diastole.
Preferably, the peptide or a homologue, analogue or derivative thereof induces
at least
about a 3% or 5% increase in systolic [Ca2+]; relative to the systolic [Ca2+];
dete~i~ed in
the absence of the peptide from a standard in vitro calcium-sensitizing assay.
Prefr~rahly,
the peptide or a homologue, analogue or derivative thereafind~aces at least
about a 3~'~'0 ~r
5% decrease in diastolic [Ca2+]; relative to the diastolic [Ca'~]; determined
in the absence
of the peptide from a standard ire vitro calcium-sensitizing assay. More
preferably, cyst~lic
[Ca2+]; is increased, or diastolic [Ca2+]; is decreased, by at least about 10%
or 15%, and still
more preferably by at least about 20%, 25%, 30%, ~~0% or ~0%, relative to the
systolic
[Ca2+]; or diastolic [Ca2+]; respectively, that is measured in absence of the
peptide in such a
standard ih vitro calcium-sensitizing assay.
Even more preferably, the administered peptide, or a homologue, analogue or
derivative
thereof, improves the efficiency of cardiac contraction. Preferably, cardiac
contraction is
enhanced by inducing at least about a 5% or 10% increase in preload-
recruitable stroke
work (PRSW) at within 0.5-1.0 hr following administration. More preferably,
cardiac
contraction is enhanced by about 15%, 20%, 30%, 40°,/0, ~0~~'~,'SS%,
60% or 70%
determined by an increase in PRSW in heart failure subjects relative to
healthy indi~riduals.
For example, the peptide, or a homologue, analogue or derivative thereof, can
be
immediately administered to a patient (e.g. i.p. or i.v.) that a~:~s suffered
or is suffea~ing from
congestive heart failure or cardiogenic shock. Such immediate administration
preferably
would entail administration of a suitable dosage of peptide to enhance RyR2
calcium
channel activity within about 1, 2, 4, 8, 12 or 24 hours, or from more than
one day to about
2 or three weeks, after the subject has suffered from heart failure such as
congestive heart
failure or cardiogenic shock.
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Relatively long-term administration of the peptide, or a homologue, analogue
or derivative
thereof, at a dosage suitable to activate RyR2 calcium channel activity will
also be
beneficial after a patient has suffered from chronic heart failure, to provide
increased
exercise tolerance and functional capacity. For example, the peptide can be
ad~~nir~istered
regularly to a patient for at least 2, 4, 6, 8, 12, 16, 18, 20 or 24 weeks, or
longer ;such as, for
example, 6 months, 1 years, 2 years, 3 years or more, after having suffered
heart failure to
promote enhanced functional capacity. An oral dosage formulation would be
preferred. for
such long-term administration.
The administration of the peptide, or a homologue, analogue or derivative
thereof, at. a
dosage that inhibits cardiac RyR2 calcium channel activity in human or animal
subjects is
not excluded, and may be appropriate, for example, in cases where transient
relaxation of
cardiac tissue is required.
Toxicity, and the therapeutic efficacy of the peptide, homologue, analogue or
derivative,
can be determined by standard pharmaceutical procedures in cell cultures or
experimental
animals, e.g., for determining the LDSO (the dose lethal to 50% of the
population) and the
EDSO (the dose therapeutically effective in 50% of the population). The dose
ratio between
toxic and therapeutic effects is the therapeutic index, and this index can be
expressed .as the
ratio LDSo/EDSO. The amino acid sequence set forth in any one of SEQ ID NOs: 1-
~7, o~° a
homologue, analogue, or derivative thereof having a high therapeutic index, is
preferred..
Whilst peptides, homologues, analogues, or derivatives, that exhibit toxic
side effects ~~r
have a high LDSO value are less desirable, such peptides may be used in
conjuncddn ~~tith a
delivery system that targets such compounds to the site of affected tissue,
thereby
minimizing potential damage to healthy tissue.
Data obtained from cell based assays and animal studies can be used in
formulating a range
of dosage of the subject peptides for use in humans. The animal models of
cardiac
hypertrophy described by Grant et al. (LTSSN 6,201,165 issued March 13, 2001)
are
particularly useful for this purpose. The dosage of peptide, homologue,
analogue, or
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derivative, lies preferably within a range of concentrations that, following
administration
by a particular route, produce a circulating concentration consistent with the
EDSO and
having little or no toxicity. The dosage may vary within this range depending
upon the
dosage form and route of administration. The dosage may also vary according to
factors
such as the disease, severity of disease, age, sex, and weight of the
individual.
For any peptide, homologue, analogue, or derivative, used in the method of the
inv er~.fion,
the therapeutically effective dose can be estimated initially frorrg cell
based assays ~r
animal models. For example, an effective dose may be fort~~ulated in animal
mt~dels to
achieve a circulating plasma concentration range that includes the ICSO (i.e.,
the
concentration of peptide or non-peptide compound that achieves a half maximal
in~abition
of symptoms) determined from cell based assays and/or in whole animals. Such
information can be used to more accurately determ.ir~e usefml doses in humans.
Suitable dosages of the peptide, or a homologue, analogue or derivative
thereof, can also
be determined by inducing heart failure in an animal model by chronic rapid
ventricular
pacing, an then infusing different concentrations of the peptide into the
right atrium at a
rate of about 3.3 mL/min and then recording the pressure-dhnension
relationships and. the
arterial pressure response to the peptide. Control experiments using lazown
calcium
sensitizers, such as those described by Marban (USSN &., l~l,I3~ issued on
Febrt~ay 2~,
2001) can also be performed. Cardiac oxygen consumption also may be measured.
Yet another aspect of the present invention is directed to a pharmaceutical
compc~siti~n
comprising a fragment of a dihydropyridine receptor polypeptide, which peptide
comprises
at least about 5 contiguous amino acid residues of the peptide set forth in
any one of SEQ
ID NOs: 1-10, or a homologue, analogue or derivative thereof together with sin
a or more
pharmaceutically acceptable carriers and/or diluents.
The therapeutic efficacy of a substance detected by the methods of the present
invention in
the treatment of cardiac dysfunction can be accomplished by those skilled in
the art using
known principles of diagnosis and treatment.
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Therapeutic compositions must be sterile and stable under the conditions of
manufacture
and storage. The peptides can be formulated as a solution, microemulsion,
liposome, or
other ordered structure suitable to high drug concentration. The carrier can
be a solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures
thereof. Proper fluidity can be maintained, for example, by the use of ~a
coating such as
lecithin, by the maintenance of the required particle size in the case
of.dispersion and by
the use of surfactants. In many cases, it will be preferable to include
isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride
in the
composition.
Enhanced absorption can be achieved by conjugation of the peptide, or a
homologue,
analogue or derivative thereof, to a lipid or liposaccharide moiety.
Prolonged absorption of the injectable compositions can be brought about by
including in
the composition an agent that delays absorption, for example, a monostearate
salt or
gelatin. Moreover, the compounds can be administered in a time release
formulation, for
example in a composition which includes a slow release or controlled release
polymer,
preferably coyprising a hydrophobic andlor amphiplailic compound. The peptides
can be
prepared with carriers that will protect the comporand against rapid
rele~as~e, such as a
controlled release formulation, including implants and microencapsulated
delivery
systems. Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl
acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and
polylactic, polyglycolic copolymers (PLG). Methods for the preparation of such
formulations are generally known to those skilled in the art.
For example, a suitable controlled-release delivery ampoule can be prepared by
dispersing
the peptide, homologue, analogue or derivative, in a bioerodable,
biodegradable poly(e-
caprolactone) polymer matrix in the melt stage, provided the peptide or
protein drug is
dispersed in a glassy matrix phase having a glass transition temperature that
is higher than
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the melting point of the poly(e-caprolactone) polymer, such as, for example, a
glassy
matrix phase produced by lyophilizing an aqueous solution of the peptide and a
suitable
thermoprotectant (e.g. trehalose, melezitose, lactose, maltose, cellobiose,
melibiose, or
raffinose) as described by Wang et al. (LISSN 6,1 X7,330).
Sterile injectable solutions can be prepared by incorporating the peptide,
homologue,
analogue or derivative, in the required amount in an appropriate solvent with
one or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
peptide or
non-peptide compound into a sterile vehicle which contains a basic dispersion
medium and
the required other ingredients from those enumerated above. In the case of
sterile powders
for the preparation of sterile injectable solutions, the prefeiTed methods of
preparation are
vacuum drying and freeze-drying which yields a powder of the active ingredient
plus any
additional desired ingredient from a previously sterile-filtered solution
thereof.
Preferably, the peptide of the invention, or a homologue, analogue or
derivative thereof, is
formulated to extend its half life following administration, particularly in
formulations for
the treatment of chronic conditions. Methods for extending the half life of
the active
peptide compound includes direct modification to reduce its proteolysis, such
as, for
example, by cross-linking or amino acid substitutions to remove sites for
known proteases.
Alternatively, half life of any active ingredient can be extended by
encapsulating it in a
suitable formulation, such as, for example, a slow release formulation.
Depending on the
route of administration, the peptide, homologue, analogue or derivative, may
be coated in a
material to protect it from the action of enzymes, acids and other natural
conditions which
may lead to its inactivation.
For example, the peptide, homologue, analogue or derivative, can be
administered to a
subject in an appropriate carrier or diluent co-administered with enzyme
inhibitors or in an
appropriate carrier such' as liposomes. Pharmaceutically acceptable diluents
include saline
and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin
inhibitor,
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diisopropylfluoro-phosphate (DEP) and trasylol. Liposomes include water-in-oil-
in-water
emulsions as well as conventional liposomes.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and
n~xt~~res
thereof and in oils. Under ordinary conditions of storage and use, these
preparations gay
contain a preservative to prevent the growth of microorganisms.
This invention is also described with reference to the following non-limiting
examples.
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EXAMPLE 1
A 20-MER PEPTIDE THAT MODULATES CARDIAC RYR2 CALCIUM
CHANNEL ACTIVITY
MATERIALS & METHODS
Materials.
Chemicals and biochemicals were from. Sigma-Aldrich. (Castle Hill, Australia).
The
DHPR II-III loop peptides (SEQ ID Nos: 1-7) were synthesized using an Applied
Biosystems 430A Peptide Synthesizer with purificati~rrr~ to >_98 % using HPLC
and mass
spectroscopy and NMR. Peptide was prepared in a ~2 .rnM stock solution in Hz0
and
frozen in 20 p.1 aliquots. Precise stock solution concer~t~wtions were
determined by Auspep
Pty Ltd using acid hydrolysis followed by a standardized PTC
(phenylthiocarbamyl)
protocol and analysed by reverse phase HPLC.
Peptides
Particular peptides used for the study were:
1. The 20-mer peptide of the II-II cytoplasmic loop of DHPR (SEQ ID NO: 2):
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys A~:g Arg Lys Met Ser Lys Gly
Le~x
2. Peptide NB (the N-terminal portion of the B segment of the I~-III loop;
Hamilton
and Ianuzzo, 1991; SEQ ID NO: 11):
Gly Leu Pro Asp Lys Thr Glu Glu Glu Lys Ser Val Met Ala Lys Lys Leu Glu Gln
Lys
3. Peptide AlS (scrambled 20-mer peptide; SEQ ID NO: 12):
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Thr Arg Lys Ser Arg Leu Ala Arg Gly Gln Lys Ala Lys Ala Lys Ser Glu Met Arg
Glu
SR vesicle Preparation.
SR vesicles were isolated from sheep heart as described by Laver et al. (1995,
J. O~Ie~~a~r.
Biol. 147:7-22).
Lipid Bilayers.
Experiments were carried out at 20°C to 25°C, essentially
according to Ahern et al. (1994,
FEBS Lett. 352:369-374) and Laver et al. (199, ~: Membr. Biol. 147:7-22).
Bilayers were
formed from phosphatidylethanolamine, phosphatidylserine and
phosphatidylcholine
(5:3:2 w/w/w) (Avanti Polar Lipids, Alabaster, Alabama) across an aperture
with a
diameter of 100 ~.m in the wall of a 1.0 ml Delrin cup (Cadillac Plastics,
Australia).
Terminal cisternae (TC) vesicles (final concentration, 10 ~g/ml) and drags
were added to
the cytoplasmic (i.e. Gis) chamber. The bilayer potential was controlled, and
single
channel activity recorded, using an Axopatch 200A amplifier (Axon Instruments,
Foster
City, CA). For experimental purposes, the cis chamber was held at ground and
the voltage
of the lumen (i.e. t>~ans) chamber controlled. Bilayer potential is expressed
in the
conventional Way aS Vcls - Vtrans~ (1~e~ cytoplasm' Vlumen)~
Bilayer solutions.
Bilayers were formed as described above and vesicles were incorporated into
the bilayer
using a cis solution containing 230mM Cs methanesulphonate (MS), 20mM CsCI,
1mM
CaCla and lOmM N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES, pH
7.4
with CsOH), and a traps solution containing 30mM Cs MS, 20mM CsCI, 1mM CaCl2
and
l OmM TES (pH7.4).
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To prevent incorporation of multiple channels into the bilayer, the cis
solution ~~uas
replaced by perfusion of the cis chamber when channel activity was observed.
The cis
perfusion solution was identical to the initial cis solution, except that the
[Ca2+' ~~Fas
3 x 10'~M, buffered using 1 mM BAPTA. CsMS (200mM) was added to the t~afas
chamber
following channel incorporation to produce symmetrical solutions.
Recordiytg sisZgle chahnel aetivity and data aa~alys~~
Bilayer potential was changed every 30s, over 2 min .follov,~ing each addition
of peptide to
the cis chamber. Control activity was recorded for 2 min after addition of
200mM CsMS
to the tans chamber. Activity was then recorded for 2 min after cis addition
of each
aliquot of peptide (six different peptide concentrations were examined in each
channel).
Peptides were perfused out of the cis chamber, and the recovery recorded.
Channels were
finally exposed to 30~M ruthenium red.
Currents were filtered at 1 lcHz (8-pole low pass Bessel, -3 dB) and digitized
at 5 kHz.
Analysis of single channel records (using Channel 2~ developed by PW Gage and
~r'1
Smith) yielded channel open probability (PD), frequency of es~enta (~o), open
times, closed
times and mean open or closed times (TO or T~) as well as meaa~. current (I').
The event.
discriminator was set above the baseline noise at ~-20% ~of the max$mum
current, rather
than the usual 50%, and so openings to both sub conductance arzd maximum
conda~.cce
levels were included in the analysis. Channel activity was analyzed over two
30s periods
of continuous activity at +40 mV and two 30s periods of continuous activity at
-~~9rnV.
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Statistical analyses.
Average data is given as mean ~ SEM. The significance of the difference
between control
and test values was tested using one sided or two sided Students T-tests, for
independent or
paired data, as appropriate. Differences were considered to be significant
when P<_0.05.
EXAMPLE 2
A 20-MER PEPTIDE THAT MODULATES CARDIAC RyR2 CALCIUM
CHANNEL ACTIVITY
RESULTS
An increase in the activity of RyRs from cardiac muscle was seen when the 20-
leer peptide
(SEQ ID NO: 2) was added to the cytoplasmic (cis) side of the channel at a
concentration
of 10-~M cis Ca2+. Single channels were identified as RyRs by their Cs+
conductance of
450 pS with bilayer potentials of +40mV or -40mV and by the ability of 30 ~.M
ruthenium red to block the channel at the end of the experiment.
Records from one experiment in which cardiac RyRs were strongly activated by
the 20-
mer peptide (SEQ ID NO: 2) are shOWll 111 Figure 2, at-40 mV, arld.in Figure
3, at +40
mV. Channel activity before addition of the peptide consisted of
briefinterrl~ltent
openings (Figure 2, panel A; Figure 3, panel A). Within 10 s of adding 65nM
peptide (SEQ
ID NO: 2), channel openings increased at-40 mV.
An increase in the frequency of events, and the appearance of very long
openings, were
accompanied by the opening of a second channel in the bilayer (Figure 2, panel
B). RyR2
channel activity at -40mV fell slightly when peptide concentration was
increased to 6.5
~,M SEQ ID NO: 2, although the activity of the second channel was more
pronounced. As
shown in Figure 2, panel C, the channel shows a high open probability (Po),
particularly at
65nM peptide (the predominant current level, O, expected if the channel was
fully open).
~n
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The records show that the increase in single channel activity was not
associated with any
change in single channel conductance.
Channel activity also increased at 65 nM 20-mer peptide (SEQ ID I°~,;0:
2) when the bilayer
potential was +40mV (Figure 3, panel B). however, the increase in channel
activity wa.s
less than that observed for the same channel at -40mV (Figure 2, panel f3).
Whilst channel activity at -40mV bilayer potential,increased at higher peptide
concentration, channel opening at +40mV began to decline at concentrations
above about
100nM peptide, and only a few brief channel openings were observed at 10~,M
peptide,
due to low conductance levels (Figure 3, panel C). Without limiting the
invention to any
one theory or mode of action, the depression of activity at +40mV, wherein
channel
openings are mostly due to sub maximal conductance, is thought to be
attributable to
binding of the peptide (SEQ ID NO: 2) to low affinity sites that axe distinct
from the sites
at which the peptide binds during channel activation. These low affinity sites
are probably
within the channel pore.
Similar activation and inhibition of cardiac RyR2 channels in lipid bilayers
was obtained in
8 out of 8 bilayer samples. Single channel analysis was not performed since
most bilayers
contained more than one channel. All samples had a ~.igher frequency of
opening at
+40mV, and at -40mV, following peptide (SEQ ID NO: 2) addition to tlxe c~.s
chamber.
Additionally, prolonged channel openings occurred at a potential of -40mV in
the presence
of peptide, compared to that detected in the absence of peptide at 10'~~M eis
Ca2+.
The mean current (i.e. the average of all data points in two 30s records, at
each potential
and each peptide concentration, divided by the number of channels seen in the
recording)
provided a measure of channel activity. Average normalized mean current is
shown as a
function of peptide concentration in Figure 4. The integer I'p/I'c is the
ratio of mean
current in the presence of peptide to mean current before peptide addition to
the chamber.
A significant increase in mean current (~2-fold ) was detected in lOnM peptide
(SEQ ID
NO: 2), at a bilayer potential of -40mV or +40mV. Mean current at -40mV
increased up to
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a further 4-fold in 50 ~M peptide (SEQ ID NO: 2). In contrast, the normalized
mean
current at +40mV doubled at l OnM peptide (SEQ ID NO: 2), remained elevated up
to
about 10~M peptide, and then fell dramatically at higher peptide
concentrations (i.e.
between about 10~,M and SO~,M peptide).
The specificity of the activation of cardiac RyR2 channels by the 20-mer
pefytide (SEQ ID
NO: 2) was also tested. At 1 ~,M cis peptide NB (SEQ ID NO: 8) or 10~,M cis
peptide NB,
no modified channel activity was observed at +40mV or -40rr~.V bilayer
potential (n=3;
Figure 4). A scrambled sequence having the same isoelectric point as the test:
20-mer
peptide but a non-homologous sequence (i.e. Peptide Al S; 'SEQ ID NO: 9)
reda.~ced cardiac
RyR2 channel activity when present at higher concentrations (i.e. 1 ~.M cis
peptide or
10~.M cis peptide) in 2 out of 2 of the other bilayers (Figure 4).
Data provide strong support that activation by the 20-mer peptide (SEQ ID NO:
2) reflects
binding to the cardiac RyR2 channel.
Without limiting the present invention in any way, the ability of the 20-mer
peptide (SEQ
ID NO: 2) and peptide A1S at higher concentrations to inhibit cardiac RyR
activity at
+40mV suggests, not unexpectedly, that the pore or its vestibule in both
cardiac and
skeletal RyRs contain a significant number of negative sitesvvhich can
interact ~Tith
positively charged residues in the peptides. The fact that inhibition persists
v,~z~ith peptide
Al S in the absence of activation provides further evidence that activation
and inhibition
depend on the 20-mer peptide (SEQ ID NO: 2) binding to twao separate sites on
the RyR2
channel.
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EXAMPLE 3
FUNCTIONAL ANALYSIS OF DHPR 20-MER FRAGMENT DERIVATIVES AND
ANALOGUES
MATERIALS & METHODS
Peptides
Four peptides were tested in this series of experiments, these being:
(i) the native DHPR 20-mer peptide (SEQ ID NO: 2);
(ii) the SEQ ID NO: 2 peptide in which Ser~87 residue 17) is replaced by an
alanine residue to produce SEQ ID NO: 8;
(iii) the SEQ ID NO: 2 peptide in which Arg6$8 (residue 18) is replaced by an
D
isomer to produce SEQ ID NO: 9; and
(iv) the SEQ ID NO: 2 peptide in which Ser68~ is mutated to alanh~.e and
Arg688
is replaced by a D isomer to produce SEQ ID NO: 10.
Measuremeaats of Ca''~ f~elease f~~or~z cardiac SR
Cardiac SR vesicles (50 ~,g of protein) were added to a cuvette, to a final
volume of 2 ml
of a solution containing (in mM): 100, KHZP04 (pH = 7); 4, MgCh; 1, Na2ATP;
0.5,
antipyrylazo III. Extravesicular [Ca2+] was monitored at 710 nm using either a
Cary 50 or
Cary 100 spectrophotometer. Identical experiments at 790 nm showed no changes
in OD
that were independent of changes in [Ca2+] which would alter the rate of Ca2+
release
measured at 710 nm. Vesicles were loaded with Ca2+ by 4 additions of 3p,1
aliquots of ~
mM CaCl2, to a final concentration of 7.5 ~,M Caz+. Thapsigargin (200nM) was
then
added to block the SR Ca2+ ATPase. Finally the peptides were added either
alone, or with
20 ~.M Ca2+ or 2 mM caffeine. The rate of Caz+ release in the presence of
thapsigargin was
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measured just before addition of the activating agents and initial rates of
Ca2+ release were
measured immediately after addition of the activating agents. The specific
Mocker of RyR
activity, 5 ~.M ruthenium red, was then added to confirm that Caz+ release wa
~ r~rough RyR
channels. The Ca2+ ionophore A23187 (3 ~,g/ml) was added at the end of the
experiment
to determine the amount of Ca2+ remaining in the SR vesicles. When Ca2+
release ~waa
complete, the Ca2+ transient following addition of the ionophore, was
indicatives of the
fraction of vesicles containing Ca2+ that could not be released through RyR
chaa3nel
(presumably vesicles from longitudinal SR that lack RyR channels). The results
~ho~ved
that only 10-20% of the cardiac SR preparation contained RyR regulated stores.
Experiments were repeated on vesicles isolated front 3 different sheep heart
preparations.
Single channel experiments.
The Ca2+ concentration in the cis solution was either buffered at 100 nM or
was adjusted to
100 ~.M by adding an CaCl2 to the cis solution in the absence of BAPTA.
EXAMPLE 4
FUNCTIONAL ANAL~'SIS OF DHPR 20-MER PEPTIDE FRAGMEN':~
DERIVATIVES AND ANALOOL~S
RESULTS
Sasmmary
The example 4 experiments include the testing of all compounds on Ca2+ release
from
cardiac SR vesicles. In addition, single cardiac RyR channels were exposed to
peptide
SEQ ID NO: 9. All peptides significantly enhanced the rates of Cat+-activated
Ca2~
release or caffeine-activated Ca~+ release. Peptides SEQ ID NOs: 9 and 107 at
high
concentrations of >30~M marginally enhanced the rate of Ca2+ release in the
absence of
activating Ca2+ or caffeine, and also enhanced both Cat+-activated Ca2+
release and
caffeine-activated Caa+ release. Peptide SEQ ID NO: 9 enhanced cardiac RyR
activity in
lipid bilayer experiments at low concentrations (1-l OnM), but blocked the
channel pore in
a voltage- and Cat+- dependent manner at +40mV at high concentrations.
Accordingly,
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under appropriate physiological conditions, the dihydropyridine receptor
fragment peptides
are able to bind to, and activate, the cardiac Caa+ release channel and Ca2+
release from
cardiac SR.
Effects of the A peptides oh e'a2+ release from cardiae SR.
The ability of the peptides defined by SEQ III NOs: 2, 8, 9 and 10 to release
Caz-~~ ~-~n~
cardiac SR, in the absence of additional activating factors, was determined.
The peptides
were added to the extravesicular solution and initial rates of Ca2+ release
immediately
following the addition of peptides were measured. Average data for the four
peptides is
shown in Fig. 5 (small symbols). A small increase in the rate of release was
seen with all
peptides at concentrations of 30~,M and SO~,M.
The effects of the peptides on Ca2+-activated Ca2+ release and caffeine-
activated Ca2+
release were examined. Ca2+-activation is the principal ih vivo mechanism of
RyR
activation during cardiac contraction. The Ca2+-activation mechanism is
responsible for
both Ca2+-activated and caffeine-activated Ca2+ release from SR vesicles,
since the major
effect of caffeine is to shift the Caz+-activation curve to much lower Ca2+
concentrations.
The resting Ca2+ concentration of 100 nM activates Ca"+ release in the
presence of
caffeine. The initial rates of Ca2+ release induced by 20 ~M Ca~'+ and 2 mM
caffesz~.e were
similar and were ~10 ,moles of Caz+ per mg of SR protein per znin in these
experig~xezats.
The effects of the peptides on Ca2+-induced Ca2+ release and caffeine-induced
Ca~+ release
were also similar and data obtained with the two methods of activation is
grouped trrgether
in the average data shown in Fig. 5. Clearly, each of the four peptides was
able to ia~crease
the Ca2+/caffeine-activated Ca2+ release from the cardiac SR vesicles when
added to the
extravesicular solution. In all experiments, Ca2+ release was terminated by
addition of
ruthenium red, indicating that release was through RyR channels. The maximum
rate of
Ca2+ release with each of the four peptides was 2 to 2.5-fold greater than
control and was
achieved at 20-30 ~M peptide. There appeared to be a biphasic action with the
four
peptides in that the rates of Ca2+ release tended to fall at higher peptide
concentrations.
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The peptides all showed similar potency in releasing Caa+ from cardiac SR and
in
enhancing Ca2+/caffeine-activated Ca2+ release. It is possible that the
release from non
Ca2+/caffeine-activated channels was too small to pick up differences
bet~treen peptides.
Without limiting the invention in any way, this similarity between the effects
of the
peptides on Ca2+/caffeine-activated Ca2+ release may have reflected the fact
that the RyR
channels were close to maximally open with the activating agents alone and
this their
capacity to be further activated by the peptides was limited.
Effects ~f peptide SEQ ID N~: 9 vaa sixagi~.~ardiac RyR cBaa~a.ael.activity.
The action of SEQ ID NO: 9 on the acti~~it~y Bangle cardiac RyR channels in
lipid bilayers
was examined. The peptide was initially added to cytoplasmic (cis) side of
cardiac RyR
channels with 100 ~.M cis Ca2+. The peptide failed to activate channels (Fig.
6).
The reduction in the activity of cardiac RyR channels induced when peptide SEQ
ID NO: 9
was added to the bilayer solution was greater at +40 mV than --40 mV and was
thus similar
to the voltage-dependent (i.e. current direction-dependent) block of skeletal
RyR channels
by peptide SEQ ID NO: 2. The reduction in activity is consistent with the
positively
charges SEQ ID NO: 9 peptide entering tlae ion channel and associating with
negative
charges within the pore. The block is ea~hanced when current flow is from cis
to tans and
carries the peptide into the pore, but .is partially reversed when current
flows from traps to
eis and tends to carry the peptide out of the pore.
A cis Ca2+ concentrati~~n of 100 nM peptide, buffered with 2 mM BAPTA v~~as
used.
Strong activation of the cardiac channels by peptide SEQ ID NO: 9 caused a 10-
20 fold
increase in relative mean current at both positive and negative potentials
(Fig. 7). There
was a significant increase in activity with only 10 nM peptide and maximum
activation
was observed with 100 nM peptide. A fall in the relative mean current at
higher peptide
concentrations (1 and 10 pM) at +40 mV was indicative of a residual blocking
action of
the peptide.
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The effects of this peptide on average single channel parameters of cardiac
RyRs was
measured and average data is shown in Fig. 8. The peptide induced an ~50-fold
increase
in the open probability -40mV and an ~10-fold increase at +40m~. This increase
in
activity was due to an 8-fold increase in the mean open time at both
potentials and an 80-
or 170-fold decrease in the mean closed times at +40 mV and -40 mV
respectively, and a
corresponding increase in the frequency of events. Thus the major effect on
the peptide on
channel gating is on the mean closed time.
Accordingly, the DHPR II-III loop SEQ ID NO: 2 peptide can activate cardiac
RyR
channels and Ca2+ release from cardiac SR vesicles. Not only does the native
peptide
release Ca2+, several other peptides with enhanced helical structure, and
hre:~erably
containing the RI~RRK sequence, exhibit the same action on Ca2+ release.
Those skilled in the art will appreciate that the invention described herein
is susceptible to
variations and modifications other than those specifically described. It is to
be understood
that the invention includes all such variations and modifications. The
invention also
includes all of the steps, features, compositions and compounds referred to or
indicated in
this specification, individually or collectively, and any and all combinations
or any two or
more of said steps or features.
The present invention is not to be limited in scope by the specific
embodiments descried
herein, which are intended for the purposes of exemplification only.
Fun.ctionally-
equivalent products, compositions and methods are clearly within the scope of
the
invention, as described herein.
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REFERENCES
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SEQUENCE LISTING
<110> The Australian National University
<120> Method of modulating the activity of calcium channels in cardiac
cells and reagents therefor
<130> 2533538/TDO
<160> 12
<170> PatentIn version 3.1
<210> 1
<211> 21
<212> PRT
<213> synthetic consensus DHPR 20-mer peptide
<220>
<221> MISC FEATURE
<222> (1)..(21)
<223> Xaa at positions 6 and 8 is any amino acid, preferably Ala or Glu;
Xaa at position 7 is any amino acid, preferably Glu or Lys; Xaa at
positions 11, 14, and 18 is Arg or Lys; Xaa at position 12 is any
amino acid, preferably Arg or Glu; Xaa at position 3~a is Met, Leu,
Ile, or Val; Xaa at position 17 is Ala or Ser; X~,a at position 18
is Lys or Arg; Xaa at position 18 is any amino acid, preferably
Gly, Thr, Ala; Xaa at position 19 is any amino acid, preferably
Leu, A1a or Asn.
<400> 1
Thr Ser Ala Gln Lys Xaa Xaa Xaa Xaa G1u Glu Xaa Xaa Arg Ser Lys
1 5 10 15
Xaa Xaa Xaa Xaa Xaa
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<210> 2
<211> 20
<212> PRT
<213> synthetic murine skeletal DHPR 20-mer peptide
<400> 2
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Arg L~~s Met
1 5 10 l5
Ser Lys Gly Leu
<210> 3
<211> 20
<212> PRT
<213> synthetic murine skeletal DHPR 20-mer peptide
<400> 3
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Arg Lys Met
1 5 10 15
Ser Lys Gly Leu
<210> 4
<211> 20
<212> PRT
<213> synthetic rabbit skeletal DHPR 20-mer peptide
<400> 4
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Arg Lys Met
1 5 10 15
Ser Arg Gly Leu
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<210> 5
<211> 20
<212> PRT
<213> synthetic rabbit cardiac DHPR 20-mer peptide
<400> 5
Thr Ser Ala Gln Lys Glu Glu G1u Glu Glu Lys Glu Arg Lys Lys Leu
1 5 10 1.5
Ala Arg Thr Ala
<210> 6
<211> 20
<212> PRT
<213> synthetic rat cardiac DHPR 20-mer peptide
<400> 6
Thr Ser Ala Gln Lys Glu Glu Glu G1u G1u Lys Glu Arg Lys Lys Leu
1 5 10 15
Ala Arg Thr Ala
<210> 7
<211> 20
<212> PRT
<213> synthetic bullfrog skeletal DHPR 20-mer peptide
<400> 7
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Lys Lys Leu
1 5 10 15
Ala Arg Ala Asn
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<210> 8
<211> 20
<212> PRT
<213> synthetic derivative of human skeletal DHPR 20-mer peptide
<400> 8
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Arg Lys Met
1 5 10 15
Ala Arg Gly Leu
<210> 9
<211> 20
<212> PRT
<213> analogue of human skeletal SHPR 20-mer peptide
<220>
<221> MISC FEATURE
<222> (1)..(20)
<223> X is the' D isomer of Arg
<400> 9
Thr Ser Ala G1n Lys Ala Lys A1a G1u Glu Arg Lys.Arg Arg Lys Cflet
1 5 10 15
Ser Xaa Gly Leu
<210> 10
<211> 20
<212> PRT
<213> analogue of human skeletal DHPR 20-mer peptide
<220>
<221> MISC FEATURE
<222> (1)..(20)
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<223> X is the D isomer of Arg
<400> 10
Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Arg Lys Met
1 5 10 15
Ala Xaa Gly Leu
<210> 11
<211> 20
<212> PRT
<213> synthetic peptide NB
<400> 11
Gly Leu Pro Asp Lys Thr Glu Glu G1u Lys Ser ~~al Met Ala Lys Lys
1 5 10 15
Leu Glu Gln Lys
<210> 12
<211> 20
<212> PRT
<213> synthetic peptide A1S
<400> 12
Thr Arg Lys Ser Arg Leu Ala Arg Gly Gln Lys Ala Lys Ala Lys Ser
1 5 10 15
Glu Met Arg Glu
<210> 13
<211> 37
<212> PRT
<213> synthetic RyR2 peptide
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<400> 13
Phe Arg Ala Glu Lys Thr Tyr Ala Val Lys Ala Gly Arg Trp Tyr Phe
1 5 10 15
Glu Phe Glu Ala Val Thr Ser Gly Asp Met Arg Val Gly Trp Ser Arg
20 25 30
Pro Gly Cys Gln Pro
